ELISpot assays employ the sandwich enzyme-linked immunosorbent assay (ELISA) technique. Either a monoclonal or polyclonal antibody specific for the chosen analyte is typically pre-coated onto a PVDF (polyvinylidene difluoride)-backed microplate. Appropriately stimulated cells are pipetted into the wells and the microplate is placed into a humidified 37° C. CO2 incubator for a specified period of time. During this incubation period, the immobilized antibody, in the immediate vicinity of the secreting cells, binds secreted analyte. After washing away any cells and unbound substances, an antibody specific for the chosen analyte is added to the wells. The detecting antibody can either be biotinylated or it may be conjugated directly to alkaline-phosphatase, horseradish peroxidase, or another enzyme. With the former, additional steps of incubating with enzyme-conjugated-streptavidin and washing are performed. Regardless of the approach, following all wash procedures a substrate solution is added. A colored precipitate forms and appears as spots at the sites of analyte localization, with each individual spot representing an individual analyte-secreting cell. The spots can be counted with an automated ELISpot reader system or manually, using a stereomicroscope.
FLUORIspot assays employ the same general procedures, except that a fluorochrome is conjugated to the detecting antibody (although biotin can be used to amplify the signal produced). This type of detecting antibody omits the need for an enzymatic detection system as described above. Instead, fluorochrome bound to the analyte of interest can be measured directly using a fluorimeter or other appropriate detection devices.
Both of these assays can only provide limited data. They can only enumerate the number of cells producing a particular antigen/analyte. Regardless of the type of assay, neither of these assays is capable of quantifying analyte secreted by the cells. It would be desirable to modify these assays in a manner that can afford analyte quantification.
The present invention is directed to overcoming these and other deficiencies in the art.